Haptoglobin (Hp) is a protein which is present in the blood of man and animals. The concentration of Hp in plasma or serum, following separation of the blood cells, varies in an individual animal and is related to the health status of the animal. Hp is one of a group of proteins the concentration of which increases dramatically following infection, inflammation or trauma. These proteins are known as the acute phase proteins.
Measurement of the concentration of Hp in plasma gives valuable diagnostic information to clinicians in human and veterinary medicine. In veterinary medicine, measurement of Hp is particularly important in assessing the health status of cattle and sheep as in these species Hp gives a particularly strong response to infection, with the concentration increasing in the circulation over 100 times. In other species, such as with man, the dog, the cat and the pig the measurement of Hp is also important as the plasma concentration increases 2 to 3 fold, which is sufficient to provide diagnostic information. Additionally in these other species a fall in Hp concentration may have value in diagnosis of haemolysis. Moreover, the measurement of Hp is of equal importance as a marker of inflammation or infection in laboratory animals such as rodents or mice.
Presently assays for Hp are based on either immunoassay or on the ability of Hp to bind to haemoglobin (Hb):
Hp in human plasma is measured in clinical biochemistry laboratories as a routine test for the acute phase response by antibody based methods with antiserum specific for human Hp. The commonest approach is by immunoturbidimetry, where the formation of a precipitate of antibody-Hp complex in solution can be measured and related to Hp concentration. However immunoturbidimetric assays are expensive when compared to routine biochemical tests as they require the preparation of a continuing supply of suitable antiserum. In addition, for use in veterinary diagnostic laboratories, tests based on antiserum to one species have to be validated for each separate species and the validation should be repeated for each new batch of antiserum used.
The original assays based on Hp-Hb binding depended on the finding that formation of the Hp-Hb complex alters the spectrophotometric absorption characteristic of Hb in proportion to the concentration of Hp in a plasma sample. But this has been replaced by making use of the innate peroxidase activity of the complex, which can be detected at a slightly acidic pH when it is proportional to the Hp content as the peroxidase activity of free haemoglobin is inhibited, allowing assays to be quantified by calibration with standard samples of Hp.
This assay is used in the majority of veterinary diagnostic laboratories which currently perform Hp assays and is preferred to immunoassay systems as it can be performed on all species with only a modest requirement for validation and it is also considerably cheaper than antibody based methods as the reagents are inexpensive. However, the automated version of this test utilises a reagent guaiacol which has a noxious odour and is not accepted by staff in many laboratories. The assay has not generally been adopted by commercial reagent suppliers probably for this reason. Attempts have been made to use other substrates for the peroxidase reaction, and while this can be achieved for manual methods, using tetra methyl benzidine (TMB), see Conner J. et al Research in Vet. Sci. (1988) 44, 82-88 a successful automated assay for Hp is still not available. This may be due to the sensitivity of TMB. It has been observed by the present inventors that serum samples without any haptoglobin (zero blanks) display a significant level of peroxidase activity capable of causing false positive results with TMB. It will be appreciated that such spurious peroxidase activity is undesirable when conducting an automated or semi-automated assay.
U.S. Pat. No. 4,695,552 describes a process for the determination of the Hp-Hb complex similar to the processes described above. However, it is identified that an acidic pH may not be sufficient to completely inactivate the peroxidase activity of free haemoglobin and that a detergent is added to substantially eliminate any peroxidase activity of the free haemoglobin. Nevertheless, there is no suggestion that components present in blood serum or plasma samples may effect the accuracy of such an assay.
It is amongst the objects of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages.
The present invention is based in part on the discovery by the present inventors that albumin and possibly other proteins present in blood samples has an undesirable xe2x80x9cperoxidase effectxe2x80x9d on assays of the type mentioned above.
The present invention therefore provides an assay for determining a level of haptoglobin in a sample, wherein the assay comprises the steps of:
a) forming a reaction mixture comprising the sample to be tested, haemoglobin and at least one reagent for reducing a peroxidase effect due to any albumin and/or any other protein(s) present in the sample,
b) allowing the sample, haemoglobin and said at least one reagent to react, so as to allow formation of an haptoglobin/haemoglobin complex; and
c) determining a level of peroxidase activity of said haptoglobin/haemoglobin complex, wherein the determination is carried out at an acidic pH sufficient to significantly reduce or substantially inactivate any peroxidase activity of uncomplexed haemoglobin.
It has been previously reported that uncomplexed haemoglobin displays a peroxidase activity at an acidic pH, but that this activity could be inactivated at pH 4.1. Makimura, S. and Suzuki, N. (1982) Jpn. J. Ven. Sci. 44, p15-21. U.S. Pat. No. 4,695,552 suggests however that a low level of peroxidase activity due to free haemoglobin may remain even at such a pH. Thus, the present assay is preferably carried out at a pH less than 4.1, for example pH 3.6-4.0, especially pH 3.8.
Typically the sample may be a blood sample generally of plasma or serum. The sample may be obtained from any animal, particularly mammalian animals, including rodentine, bovine, ovine, canine, feline, porcine and equine animals, as well as primates including humans. The sample may also be other body fluids such as milk, or ascitic fluid, or even in vitro incubation medium.
The sample may require to be diluted, if the haptoglobin concentration in the sample is above about 2 g/l, since the concentration of haptoglobin may not be accurately determined due to the non-linearity of a standard plot above such concentrations. However, the skilled addressee will readily understand that assay protocols could be developed with increased assay linearity to obviate any requirement for dilution. For example, a smaller sample volume may be used in appropriate circumstances.
The haemoglobin may be of the same origin as the animal from which the blood sample is being tested. That is, if the animal to be tested is bovine in origin, bovine haemoglobin may be employed. However, advantageously it has been found that the assay may be performed using haemoglobin of a species different from the species where the sample comes from. Thus the assay may be performed on a great variety of species while only utilising a single source of haemoglobin. Preferably also the haemoglobin is met-haemoglobin, although optionally oxy-haemoglobin may be used.
Serum and plasma from all species contains albumin, so this protein will be present in the sample to be tested at concentrations of up to 40 g/l. The present inventors have found through assessment of the best medium to use as a zero sample, for running as a blank, that there is significant interference with the assay from albumin. This interference has been detected at albumin concentrations of 1, 5, or 10% (10, 50, 100 g/l).
It has been observed that albumin does not have an innate peroxidase activity. Without wishing to be bound by theory it is thought that albumin and possibly other proteins present in blood serum, plasma or other samples to be tested may be binding to haemoglobin or possibly haematin released from the haemoglobin and preserving a peroxidase activity even at a pH (eg. pH 3.6-4.0) which would generally eliminate any free haemoglobin peroxidase activity. Moreover, this may be a reason why automated spectrophotometric assays for haptoglobin have hitherto not generally been adopted, due to the inability to obtain a zero level of peroxidase activity where haptoglobin is know to be absent. Previously this xe2x80x9cperoxidase effectxe2x80x9d may have inadvertently or accidentally been minimised due to a large dilution of the sample (eg. 500 fold) or by the use of an insensitive chromogen eg. guaiacol.
The present invention however reduces the requirement for a large dilution of the sample and allows more sensitive chromogens to be employed, by utilising at least one reagent for reducing a peroxidase effect due to any albumin or any other proteins present in the sample.
Said at least one reagent for reducing a peroxidase effect due to any albumin or any other protein(s) present in the sample may be independently selected from a) a reducing agent effective against disulphide bonds; b) a protein binding inhibitor and/or c) a chaotropic agent.
Preferably two or more of the above mentioned reagents are used to reduce any xe2x80x9cperoxidase effectxe2x80x9d. However, the skilled addressee will appreciate that an excess of any of the above mentioned reagents will cause inhibition of the haptoglobin/haemoglobin reaction which must be avoided. For example assays performed utilising 8-anilino-1-naphthylene sulphonic acid (ANS) as the protein binding inhibitor and dithiothreitol as the reducing agent effective against disulphide bonds at concentrations of about 10 mmol/l and 4 mmol/l respectively result in almost complete inhibition of haptoglobin/haemoglobin complex peroxidase activity. Thus, the skilled addressee through appropriate controls may ensure that only the spurious xe2x80x9cperoxidase effectxe2x80x9d is reduced without substantially effecting the peroxidase activity of the haptoglobin/haemoglobin complex.
Conveniently the reducing agent effective against disulphide bonds may be dithiothreitol, dithioerythritol, cysteine, mercaptoethanol, glutathione, 4,4xe2x80x2-dithiodipyridine or 5,5xe2x80x2-dithio(2-nitrobenzoic acid).
Typically the protein binding inhibitor may be ANS, protoporphyrin, bilirubin, taurodeoxycholic acids (bile salts), dicoumarol or 2-mercaptobenzothiazole.
Typically the chaotropic agent may be guanidine hydrochloride, potassium thiocyanate or sodium chloride.
Preferably also a detergent is included in the assay. It was initially thought by the present inventors that the use of a detergent was important for reducing the peroxidase effect due to any albumin or other protein present in the sample. While a detergent may appear to have such an effect, it has now been observed that the use of a detergent may be important in ensuring that other components of the assay remain solubilised when conducting the assay. Moreover only low concentrations of detergent are preferred since it appears that high concentrations (eg. 25 g/l) increase the apparent peroxidase effect. Thus, preferably a detergent is added to the reaction mixture at a total concentration of less than 20 g/l, more preferably less than 10 g/l.
Typically the detergent may be a non-ionic surfactant such as polyoxyethylene sorbitol esters (eg. Tween 20, 40, 60, 80 etc.); polyoxyethylene-p-t-octyphenol (eg. Triton X-45, X-100, etc.); and/or polyoxyethylene (PEG) alcohols (eg. Brij 35, 36, etc.), or ionic surfactant such as sodium dodecyl sulphate, CHAPS and cetrimide.
Typically haptoglobin present in the sample and added haemoglobin are allowed to react for less than 20 mins, preferably less than 10 mins and most preferably less than 5 mins.
Once said haptoglobin/haemoglobin complex has formed it is possible to detect this by way of an innate peroxidase activity of the complex whereas free haemoglobin peroxidase activity is substantially inhibited by the acid pH of the assay. A level of peroxidase activity may then be correlated with a level of haptoglobin in a sample by reference to a standard curve genera ted using known concentrations of haptoglobin.
Typically said peroxidase activity may be detected by use of a chromogen and hydrogen peroxide wherein peroxidase activity results in a colour change of the chromogen which may be detected spectrophotometrically at a particular wavelength. Such peroxidase detection using chromogenic substrates is well known in the art for assaying haemoglobin levels, glucose levels and previously for haptoglobin levels when the influence of albumin on the assay was unknown, see for example Bauer, K. J. Clin. Chem. and Clin. Biochem. (1981) Vol. 19 pp971-976; Reijic, R. et al, Clin. Chem. (1992) Vol. 38 pp522-525 and Conner, J. G. and Eckersall, P. D. Research in Vet. Sci. (1988), 44, pp82-88.
As well as the chromogenic substrates mentioned in the above papers (ie. 4-amino phenozone, 2-amino-4-hydroxybenzenesulfonic acid (AHBS) and tetra methyl benzidine (TMB)), it will be immediately evident to one skilled in the art that other chromogenic substrates such as O-phenylene diamine dihydrochloride, O-dianisidine, Na-2-OH-3-5-dichlorobenzene-sulphonate, 2,2xe2x80x2-azino-di(3ethylbenzthiazoline-6-sulphonic acid (ABTS), 4-aminoantipyrine chromotropic acid optionally in co-reaction with 8, anilino-1-naphthylene sulphonic acid (ANS) and 4-iodophenol may be used in the present assay.
A preferred assay according to the present invention for determining a level of haptoglobin in a blood sample, comprises the steps of:
a) forming a reaction mixture comprising the sample to be tested, haemoglobin and a detergent, a reducing agent effective against disulphide bonds or a chaotropic agent and a protein binding inhibitor;
b) allowing the components of the mixture to react, so as to allow formation of an haptoglobin/haemoglobin complex; and
c) determining a level of peroxidase activity of said haptoglobin/haemoglobin complex using a chromogenic substrate and spectrophotometric means, wherein the determination is carried out at an acidic pH sufficient to significantly reduce or substantially inactivate any peroxidase activity of uncomplexed haemoglobin.
A particularly preferred assay according to the present invention comprises the protein binding inhibitor/chromogenic co-reactant 8-anilino-1-naphthylene sulphonic acid (ANS) along with the chromogenic substrate 4-aminoantipyrine and phenol. It has been observed that ANS can supplement phenol in the co-oxidation reaction of hydrogen peroxide with 4-aminoantipyrine forming a blue chromogen which absorbs at 600 nm and has a greater absorbance than a red chromogen produced in the same conditions with phenol alone as co-reactant. Combination of ANS with 4-aminoantipyrine has been described previously for the determination of peroxide (see for example Chung et al, (1993), Talanta 40, p981-988).
The present invention also provides a kit for use in an haptoglobin assay according to the present invention wherein the kit comprises
i) Haemoglobin;
ii) at least one reagent for reducing an effect any albumin may impart to the assay, wherein said at least one reagent is independently selected from a) a reducing agent effective against disulphide bonds, b) a protein binding inhibitor and/or c) a chaotropic agent; and
iii) a chromogen for use in determining a level of peroxidase activity.
The kit may also comprise further components such as a buffer to maintain the desired pH for the assay, a detergent and/or standards of serum with known haptoglobin concentrations. It will be immediately evident to the skilled addressee that the components of the kit may be used to perform the haptoglobin assay with equipment such as test tubes or micro-titre plates and a spectrophotometer, as well as in automated biochemistry analysers as described herein.